1. Field of the Invention
The present invention relates to an apparatus to perform sampling in chromatographic systems with very small amounts of liquid sample, said apparatus being particularly applicable to high resolution gas chromatographic systems with cold injection, using capillary or micropacked columns, to high resolution liquid chromatographic systems, or to thin-layer chromatographic systems. Use of this apparatus makes it possible to perform controllable and reproducible sampling on very small amounts of sample, with values unattainable through the techniques usually employed for liquid sampling in chromatographic systems and particularly using micro-syringes or pipettes.
2. Description of the Prior Art
For all chromatographic systems problems arise for injecting in a volumetrically controlled and reproducible way very small quantities of sample into a chromatographic system. In gas chromatographic systems with cold injection, when a cold on-column or a cold splitless injection (according for example to the method described in U.S. Pat. No. 4,383,839) are used, it has been foreseen the necessity to inject small sample amounts in order to reduce or eliminate the problems created by a high content of diluting solvent (flooding effect, partial solvent trapping effect etc). But also when cold split injection is considered a small sample is advantageous due to the fact that it requires a lower splitting ratio and then errors or discriminations generally arising at high splitting ratios are avoided (see for example K. Grob Jr. and H. P. Neukom--Journal of H R C & C C Vol. 2 September 1979; 563-569). The modern tendency to use small bore capillary columns also requires the injection of small sample sizes unachievable with the dosing systems used today.
In thin-layer chromatography the use of very small sample volumes is especially required (A. Zlakis and R. E. Kaiser: HPTLC Elsevier Scientific Publishing Company--Institute of Chromatography Bad Durkheim 1977; 85-94).
Both these injection systems use syringes, microsyringes or pipettes as will be described later on.
The high resolution liquid chromatography systems usually use injectors with sampling valves having a loop wherein the sample is loaded for instance by means of a syringe. The loop is then connected with the column and with a source of eluting liquid solvent under pressure, to force the sample through the column.
In this case too, small volumes of sample are preferred and often imposed by the characteristic of the column. The modern trend is the use of microbore and capillary packed columns. Of course in all described injection systems it is necessary to obtain not only small sample quantities, but small sample quantities exactly measured and injected in a perfectly reproducible manner.
The microsyringes used in chromatography are generally of the type with calibrated body (capable of sampling amounts ranging from 0.2 to 10 microliters) or of the type with calibrated needle, where the piston penetrates into the needle. The latter microsyringes are capable of handling smaller quantities of samples, in a reliable and reproducible way, but only within certain limits, in particular with lower limits of about 100-200 nanoliters. Below this limit, the high surface tension of the liquid and the relatively reduced speed of the piston movement do not allow the drop, which has formed at the needle end, to fall from it, considering the reduced diameter of the outlet nozzle of the needle. Precision is moreover negatively affected by poor sealing between piston and calibrated needle. Another known system is the sampling system commonly used in the laboratory and named "pipette system", in which a calibrated tubing is filled with a liquid to be transferred by filling the pipette due to capillary forces or by sucking it into the tubing. The liquid amount placed in the tubing is retained in the tubing by the capillary forces or by closing one end when liquid aspiration has been carried out. Then, an injection of the liquid is made by opening said end or pushing the liquid by the carrier gas. This sampling method or transfer method of determined amounts of liquid is well known and has been used in gas chromatography too, but however only for quantities usually measurable in a rather rough way. The literature reports a lower limit of 25-50 nanoliters (see R. Kaiser--Gas Phase Chromatography--Vol. I pp. 90-95--Butterworths 1963--London) but thise limits are difficult to reach and anyhow require small tubes filled exclusively due to capillary forces. This implies that the volume of liquid injection is difficult to control and reproduce.
Therefore it can be considered that, of course according to the nature of the liquid substance to be sampled, a lower limit exists, generally between 50 and 200 nanoliters, below which it is not possible to go in reliable and reproducible manner using microsyringes or micropipettes.
The above mentioned quantitive limitations, however, are such that the operator is often forced to perform accessory operations imposed by the relatively high quantities of sample that has to be introduced into the chromatographic system. In particular, sometimes the sample must be diluted in a dilution ratio which is oftern very high (of the order of 1:10000 or more), with an operation which may involve difficulties in the exact analytical determination of the sample and in that it can introduce discriminations or variations in the sample original conditions.
In other cases, a splitting operation is necessary, that means the elimination of a high percentage of the quantity fed to the injector, before its introduction into the column, which operation may involve even higher risks of discriminations especially as above said, with hight split ratios. Between the known injection systems, Kaiser, Gas phase Chromatography, Vol. 11, pags. 59-62, discloses methods and apparatus for sample injection into gas chromatographic systems. Two particular types of injection devices are shown in FIGS. 26 and 27 of the reference. Both the injection devices described by Kaiser are versions of a micropipette type device. They are designed to be filled by capillarity with an uncontrollable quantity of liquid, which is then injected in its entirety into the evaporation area of sampling system. Sample sizes are in tenth of microliter range, roughly two orders of magnitude larger than the size of sample which can be obtained according to the present invention.
The device of FIG. 27 of Kaiser is quite similar to that of FIG. 26. Again, the sample size is determined in the filling operation. This device is also capable of handling solids which can be melted to give a homogeneous liquid.
Taft et al., U.S. Pat. No. 3,366,149, discloses a system for injecting samples which is particularly useful for injecting larger samples for preparative gas chromatography. In this system, a constant pressure is maintained on the surface of a liquid in a sample container communicating with a heater through a valve. The valve is controlled by a solenoid pulser, which operates to open the valve for a controllable time interval.
This reference is relied upon for its showing of pulse flow. However, the pulse operates on the valve, and the reference does not show a pressure pulse operating on a liquid whose flow is not constrained by a valve. Rather than operating to inject samples in the picoliter or nanoliter range, the system of Taft operates to inject samples of the order of milliliters. The system is not designed to deal with very small sample sizes, and operates on an entirely different principle.
It will be seen from the above comparison that the prior art sample injection devices operate by controlling sample size in the filling step and injecting the entire contents of the filled injection system using pressure in a different way from the way in which it is employed in the present invention. Even a combination of the cited references would only lead the skilled art worker to a pipette of the type shown in FIG. 26 or 27 of Kaiser which, however, is actuated by a pulsed valve. This type of device is typical of a titration burette which commonly delivers minimum sample quantities of 0.1-0.2 milliliters, and is unable to deliver samples in the picoliter or nanoliter volume range.